Craniofacial defects can occur as a result of abnormalities in neural crest (NCC) specification, migration and differentiation. NCCs are a transient population of migratory cells that undergo an epithelial-to-mesenchymal transition (EMT) to acquire motility, migrate throughout the whole embryo and then differentiate into a diverse range of cell types including peripheral nervous system ganglia and neurons, cartilage, connective tissue and bone. When misregulation occurs within NCC gene regulatory networks, the coordinated processes of EMT can be perturbed, leading to abnormalities in NCC development. Aberrant EMTs in other cell types can also induce cancer metastasis or organ fibrosis. As such, NCCs provide an outstanding in vivo model to study the molecular mechanisms underlying both developmental and disease-related EMTs. Our laboratory's long-term goal is to elucidate the molecular basis of cranial NCC development to better understand pathways regulating NCC induction, EMT/migration and subsequent differentiation. To contribute to this goal, we propose to investigate the mechanisms promoting the disassembly of premigratory NCC adherens junctions to facilitate cadherin switching, a common molecular event that underlies EMTs in NCCs and other cell types. In the chick midbrain, N-Cadherin (N-Cad) and Cadherin6B (Cad6B) are downregulated prior to EMT, and this regulation is critical as prior studies have shown that ectopic overexpression or knockdown of cadherin levels significantly impacts NCC EMT and migration. The spatio-temporal profiles of N-cad and Cad6B proteins indicate that cadherin downregulation occurs within a narrow developmental window that cannot be explained solely by transcriptional repression. We hypothesize that post-translational proteolytic processing depletes cadherin levels in premigratory NCCs to facilitate their EMT and subsequent migration. The Specific Aims of this application are to 1) Identify the molecular pathway responsible for the in vivo proteolytic cleavage of N-Cad and Cad6B in the chick midbrain and 2) Determine the role of proteolytic processing by candidate proteases in cranial NCC development. In Aim 1, we will genetically manipulate candidate protease levels in vivo to affect N-cad and Cad6B proteolysis and delineate subsequent effects on cadherin proteins and their localization. In Aim 2, we will investigate how in vivo perturbation of candidat protease activity prior to EMT impacts NCC EMT, migration, ganglia formation and gene expression within the premigratory and/or migratory gene regulatory networks. These studies will take a whole organism approach to provide insight into how proteolytic processing of substrates such as N-cad and Cad6B contributes to proper EMT and regulates cranial NCC development. In the long term, our results will facilitate the development of novel screening, diagnosis, and treatment modalities and/or illuminate new therapeutic targets to combat aberrant EMT-associated human diseases, syndromes and cancers.